Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental agent with the goal of inhibiting disease. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive antioxidant, derived from tea, that possesses remarkable cancer chemopreventive properties. Most studies with this agent are performed with cancer cells, however, it is equally important to understand whether these chemopreventive effects extend to normal pre-cancerous cells. Our initial studies show that EGCG increases normal keratinocyte differentiation, as assessed by EGCG-dependent up-regulation of involucrin (hINV) promoter activity, and increased cornified envelope formation. The hINV gene provides an ideal tool for studying the mechanism whereby EGCG regulates keratinocyte differentiation. The EGCG-associated response is mediated via an AP1 site within the hINV promoter and appears to involve a MAPK signaling cascade. Based on these results, we hypothesize that EGCG acts to prevent cancer development by promoting keratinocyte differentiation via an AP1 factor-dependent mechanism. The experiments outlined in this proposal are designed to shed light on the mechanism whereby EGCG stimulates keratinocyte differentiation. The ultimate goal is to better understand how green tea prevents skin cancer. Our first goal is to characterize the mechanisms whereby EGCG promotes an AP1-factor-dependent increase in hINV gene expression. To achieve this goal, we will measure EGCG effects on AP1 factor levels, AP1 factor/DNA interaction at the AP1 site, and AP1 factor phosphorylation state. Our second goal is to determine whether EGCG influences the p38 MAPK cascade that regulates hINV gene expression. This will be accomplished by examining the ability of dominant-negative kinases and pharmacologic inhibitors to modulate the EGCG-dependent regulation. Our third major goal is to evaluate whether EGCG can counter a pro-carcinogenic challenge - treatment with UVB. Fourth, it is important to affirm that EGCG has pro-differentiation promoting properties in epidermis. We hypothesize that EGCG will enhance keratinocyte differentiation in vivo. We will test this hypothesis using a unique series of transgenic mice that encode varying length of the hINV promoter. These studies will make it possible to study the mechanism of EGCG-dependent in vivo regulation. The central goal of this proposal is to gain new information regarding the mechanism of EGCG action. It is hoped that these studies lead to the design of new strategies for enhancing the effectiveness of these agents.